Our ongoing investigation of the functional alteration of bone marrow stromal cells, as an essential regulatory component of the marrow microenvironment, following VP-16 exposure has demonstrated that VP-16 exposure resulted in dysreguation of stromal cell expression of (1) stem cell factor (SCF), (2) stromal cell derived factor-1 (SDF-1), and (3) vascular cell adhesion moiecule-1 (VCAM-1). All of these proteins represent obligatory components of hematopoeisis. The working hypothesis for the currently active study is that clinical use of VP-16 chemotherapy results in inefficient hematopoietic reconstitution due to reduced homing of transplanted progenitor cells to the marrow, and failure of these cells to interact appropriately with stromal elements of the hematopoietic microenvironment. The request for a supplement to this ongoing investigation is to extend our understanding of the consequence of chemotherapy-induced alterations of the bone marrow [unreadable] microenvironment, to include its effects on the survival and differentiation of human embryonic stem cells. ES cells potentially provide an alternative source from which hematopoietic reconstitution can be derived. However, this potential requires an understanding of the stromal cell contribution to ES cell survival and differentiation, and the influence of a "chemotherapy damaged" microenvironment on these processes. It is likely that ES cells will be required to establish in a bone marrow that has been altered either during the course of "standard" treatment, or in preparation for transplantation. The studies outlined in this proposal will test the hypothesis that chemotherapy- induced reduction of stromal cell VCAM-1 will disrupt the ability of stromal cells to regulate ES cell viability and differentiation. The experiments proposed will take advantage of novel stromal cell lines developed as part of the original R01 which were engineered to constitutively express human VCAM-1, even in the presence of dose escalated chemotherapy. These unique feeder cell layers will allow focused investigation of the effect of VCAM-1 on ES cell survival and differentiation pathways. In addition, comparisons of control and chemotherapy-treated primary human stromal cell feeder layers will allow a broader evaluation of the differential support of ES cells on "healthy" and treated stroma using VP-16 as a model drug for pilot studies. In combination, these experiments will contribute to a better understanding of stromal cell signaling during ES cell development, and the factors that must be considered when proposing their use for hematopoietic reconstitution in the bone marrow microenvironment. [unreadable] [unreadable]